The mitochondrial phosphate transport protein (Wohlrab, H. (1980) J. Biol. Chem. 255, 8170-8173), which co-purifies with the adenine nucleotide translocase, has been isolated with a modified procedure resulting in an increased yield and in a preparation that is stable to storage in liquid nitrogen. The phosphate transport protein was incorporated into liposomes (phosphatidylethanolamine/phosphatidylcholine/phosphatidic acid, 2.75:1:1), and phosphate/phosphate exchange rates were determined. With pHe (extraliposomal) = pHi (intraliposomal) = 7.2, we found a Km of 2.5 mM independent of [(Pi)i] and a Vmax of 12 mumol/min . mg of protein. Parallel phosphate transport experiments were carried out with liposomes containing phosphate transport protein isolated from mitochondria inhibited by N-ethylmaleimide. Phosphate transported unidirectionally [pHe = pHi = 6.8; = 1.0 mM, (Pi)i = 0 mM] reaches a maximum at 30 s and is 0.13 and 0.05 mumol/mg for active and inhibited protein, respectively. At pHe = 6.8 and pHi = 8.0, the respective amounts are 0.45 and 0.05. At pHe = 8.0, the uptake becomes the same with active and inhibited protein (pHi = 6.8, 0.15 and 0.14; pHi = 8.0, 0.25 and 0.20). At all the above pH values, about the same uptake is observed with liposomes prepared without protein as those prepared with inhibited protein. The initial rate of protein catalyzed unidirectional flux [(Pi)e = 1.0 mM, pHe = 6.8; (Pi)i = 0 mM, pHi = 8.0) is 2 mumol/min . mg of protein or 8 mumol/min . mg of phosphate transport protein estimated without the adenine nucleotide translocase.